[show abstract][hide abstract] ABSTRACT: Our ability to guide cells in biomaterials for in vivo bone repair is limited and requires novel strategies. Short-interfering RNA (siRNA) allows the regulation of multiple cellular pathways. Core binding factor alpha 1 (Cbfa1) and hypoxia-inducible factor 1 (HIF-1) pathways can be modulated to direct bone formation via siRNA against guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (siGNAS1) and prolyl hydroxylase domain-containing protein 2 (siPHD2), respectively.
We determined whether the administration of siGNAS1 and siPHD2 in mesenchymal stem cells (MSCs) promotes osteogenic phenotype, the dose-dependent effects of siGNAS1 on MSC differentiation to osteogenic phenotype, and whether the two siRNAs promote bone formation in vivo.
siRNAs were administered to MSCs at Day 0, and protein expression of bone-specific markers was assessed at Days 1, 2, and 4 (n = 3/group/time point). In an in vivo model using seven sheep, chambers containing silk fibroin-chitosan (SFCS) scaffolds with siRNA were implanted over the periosteum and harvested at Days 7, 21, 36, and 70 (n = 4/group/time point, except at Day 70 [n = 2]) to assess bone formation.
siGNAS1 promoted collagen I and osteopontin expression, whereas siPHD2 had no effect in vitro. Dose-dependent effects of siGNAS1 on ALP expression were maximal at Day 1 for 10 μg/mL and Day 4 for 100 μg/mL. In vivo, by Day 70, mean bone volume increased compared to Day 7 for siGNAS1-SFCS (47.8 versus 1.8 mg/mL) and siPHD2-SFCS (61.3 versus 1.5 mg/mL).
Both siPHD2 and siGNAS1 support bone regeneration in vivo, whereas only siGNAS1 regulates bone phenotype in MSCs in vitro.
Clinical Orthopaedics and Related Research 07/2012; 470(9):2541-53. · 2.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human acellular dermal matrix (HADM) and non-cross-linked porcine acellular dermal matrix (ncl-PADM) are clinically useful for complex ventral hernia repair. Direct comparisons between the two in vivo are lacking, however. This study compared clinically relevant early outcomes with these bioprosthetic materials when used for ventral hernia repair.
Seventy-two guinea pigs underwent inlay repair of surgically created hernias with HADM (n = 37) or ncl-PADM (n = 35). Repair sites were harvested at 1, 2, or 4 weeks postoperatively. Adhesions were graded and quantified. Mechanical testing and histologic and immunohistologic (factor VIII) analyses of cellular and vascular infiltration were performed.
No infections or recurrent hernias occurred. No difference was observed in mean adhesion surface area or tenacity between groups. Mean cellular infiltration (p < 0.002, weeks 1 and 4; p < 0.006, week 2) and vascular infiltration (p < 0.0003, week 1; p < 0.0001, weeks 2 and 4) were greater in HADM. Ultimate tensile strength at the implant-musculofascia interface increased over time with both materials, but no difference was observed at 4 weeks. The mean ultimate tensile strength of explanted ncl-PADM itself was consistently greater than that of HADM. The elastic modulus (stiffness) did not differ between groups at the interface but was greater in explanted ncl-PADM (p < 0.0001, weeks 1 and 2; p < 0.02, week 4).
Both HADM and ncl-PADM become infiltrated with host cells and blood vessels within 4 weeks and have similar musculofascia-bioprosthetic interface strength. However, HADM has greater cellular and vascular infiltration. Longer-term studies will help determine whether later differences in material strength, stiffness, and remodeling affect hernia and/or bulge incidence.
Plastic and reconstructive surgery 06/2011; 127(6):2321-32. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The purpose of this study was to investigate the potential of silk fibroin and chitosan blend (SFCS) biological scaffolds for the purpose of cartilage tissue engineering with applications in tracheal tissue reconstruction. The capability of these scaffolds as cell carrier systems for chondrocytes was determined in vitro and cartilage generation in vivo on engineered chondrocyte-scaffold constructs with and without a perichondrium wrapping was tested in an in vivo nude mouse model. SFCS scaffolds supported chondrocyte adhesion, proliferation, and differentiation, determined as features of the cells based on the spherical cell morphology, increased accumulation of glycosaminoglycans, and increased collagen type II deposition with time within the scaffold framework. Perichondrium wrapping significantly (P<0.001) improved chondrogenesis within the cell-scaffold constructs in vivo. In vivo implantation for 6weeks did not generate cartilage structures resembling native trachea, although cartilage-like structures were present. The mechanical properties of the regenerated tissue increased due to the deposition of chondrogenic matrix within the SFCS scaffold structural framework of the trachea. The support of chondrogenesis by the SFCS tubular scaffold construct resulted in a mechanically sound structure and thus is a step towards an engineered trachea that could potentially support the growth of an epithelial lining resulting in a tracheal transplant with properties resembling those of the fully functional native trachea.
[show abstract][hide abstract] ABSTRACT: Porcine acellular dermal matrices (PADMs) have been used clinically for abdominal wall repair. The newer non-cross-linked PADMs, however, have not been directly compared with cross-linked PADMs. We hypothesized that chemical cross-linking affects the biologic host response to PADMs used to repair ventral hernias.
Fifty-eight guinea pigs underwent inlay repair of surgically created ventral hernias using cross-linked or non-cross-linked PADM. After animals were sacrificed at 1, 2, or 4 weeks, the tenacity of and surface area involved by adhesions to the repair sites were measured. Sections of the repair sites, including the bioprosthesis-musculofascia interface, underwent histologic analysis of cellular and vascular infiltration plus mechanical testing.
Compared with cross-linked PADM repairs, non-cross-linked PADM repairs had a significantly lower mean tenacity grade of adhesions at all timepoints and mean adhesion surface area at week 1. Mean cellular and vascular densities were significantly higher in non-cross-linked PADM at all timepoints. Cells and vessels readily infiltrated into the center of non-cross-linked PADM, but encapsulated cross-linked PADM, with a paucity of penetration into it. Mechanical properties were similar for the two PADMs (in isolation) at all timepoints; however, at the bioprosthesis-musculofascia interface, both elastic modulus and ultimate tensile strength were significantly higher at weeks 1 and 2 for non-cross-linked PADM.
Non-cross-linked PADM is rapidly infiltrated with host cells and vessels; cross-linked PADM becomes encapsulated. Non-cross-linked PADM causes weaker adhesions to repair sites while increasing the mechanical strength of the bioprosthesis-musculofascia interface at early timepoints. Non-cross-linked PADM may have early clinical advantages over cross-linked PADM for bioprosthetic abdominal wall reconstruction.
Journal of the American College of Surgeons 09/2010; 211(3):368-76. · 4.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-cross-linked porcine acellular dermal matrices have been used clinically for abdominal wall repair; however, their biologic and mechanical properties and propensity to form visceral adhesions have not been studied. The authors hypothesized that their use would result in fewer, weaker visceral adhesions than polypropylene mesh when used to repair ventral hernias and form a strong interface with the surrounding musculofascia.
Thirty-four guinea pigs underwent inlay repair of surgically created ventral hernias using polypropylene mesh, porcine acellular dermal matrix, or a composite of the two. The animals were killed at 4 weeks, and the adhesion tenacity grade and surface area of the repair site involved by adhesions were measured. Sections of the repair sites, including the implant-musculofascia interface, underwent histologic analysis and uniaxial mechanical testing.
The incidence of bowel adhesions to the repair site was significantly lower with the dermal matrix (8 percent, p < 0.01) and the matrix/mesh combination (0 percent, p < 0.001) than with polypropylene mesh alone (70 percent). The repairs made with the matrix or the matrix/mesh combination, compared with the polypropylene mesh repairs, had significantly lower mean adhesion surface areas [12.8 percent (p < 0.001), 9.2 percent (p < 0.001), and 79.9 percent] and grades [0.6 (p < 0.001), 0.6 (p < 0.001), and 2.9]. The dermal matrix underwent robust cellular and vascular infiltration. The ultimate tensile strength at the implant-musculofascia interface was similar in all groups.
Porcine acellular dermal matrix becomes incorporated into the host tissue and causes fewer adhesions to repair sites than does polypropylene mesh, with similar implant-musculofascia interface strength. It also inhibits adhesions to adjacent dermal matrix in the combination repairs. It has distinct advantages over polypropylene mesh for complex abdominal wall repairs, particularly when material placement directly over bowel is unavoidable.
Plastic and reconstructive surgery 11/2009; 125(1):167-76. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reconstruction of a critical size bone defect in the head and neck after trauma or tumor resection remains challenging. While certain defects, such as isolated orbital floor fractures, may be reconstructed with alloplastic biomaterials, larger defects or those involving load bearing bones usually require autologous tissue reconstruction. Vascularized bone free flaps remain the gold standard for large bone defects of the head and neck. These are generally lengthy, complicated, multi-step procedures that require subspecialty expertise to assure optimal outcomes.1 Invariably any procedure where autologous bone is harvested carries with it donor site morbidity.2 To spare the patient this additional morbidity and avoid potential complications associated with the harvest of this tissue, an alternative source for bone that would be sufficient to fill a critical-sized defect is needed.
Tissue Engineering Part A 10/2009; 15(9):2717-25. · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Silk fibroin-chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 + or - 4.8% at 15 min, increasing to 92.8+/-1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47+/-2.7 microm h(-1) and a mean persistence time of 41.4 + or - 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC-ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p<0.0001) for ASCs seeded on SFCS (69.0 + or - 9.0 kPa) than on glass (6.1 + or - 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p<0.05) for ASC seeded on SFCS (0.42 + or - 0.02 fibers microm(-1)) than on glass-seeded controls (0.24 + or - 0.03 fibers microm(-1)). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell-cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.
[show abstract][hide abstract] ABSTRACT: Regeneration of well-vascularized tissue for reconstruction of tumor-resected-defects requires regenerative and tissue conductive biomaterials. Two biologically derived materials, silk fibroin (SF) and chitosan (CS) were blended to create scaffolding for critical-sized bone regeneration via translation of a clinical approach in an in vivo sheep model. Periosteum was harvested from the sheep ribs and engrafted at an ectopic location over the latissimus dorsi muscle in order to provide precursor cells to the SFCS scaffolding of defined geometry. The regeneration of bone was compared with the bone chips (positive control) and empty space (negative controls) by assessing bone volume, bone mass density, and bone morphology over a period of 84 days. Ectopically grafted periosteum retained its ability to provide a cell source to the adjacent SFCS scaffold, which supported regeneration of bone of critical-size. Regeneration of bone was verified by osteocalcin expression and microCT imaging. SFCS scaffold that contained no growth factors or other bone inductive moiety was bone conductive and produced a volume of bone similar to the clinically relevant bone graft control.
Tissue Engineering Part A 03/2009; · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biologically derived nanoparticles (<100 nm) were fabricated for local and sustained therapeutic curcumin delivery to cancer cells. Silk fibroin (SF) and chitosan (CS) polymers were blended noncovalently to encapsulate curcumin in various proportions of SF and CS (75:25, 50:50, and 25:75 SF:CS) or pure SF at two concentrations (0.1% w/v and 10% w/v) using the devised capillary-microdot technique. Curcumin-polymer conjugates were frozen, lyophilized, crystallized, suspended in phosphate-buffered saline for characterization, and tested for efficacy against breast cancer cells. All nanoparticle formulations except 0.1% w/v 50:50 SFCS were less than 100 nm in size as determined with the transmission electron microscopy. The entrapment and release of curcumin over eight days was highest for SF-derived nanoparticles as compared to all SFCS blends. The uptake and efficacy of SF-coated curcumin was significantly higher (p < 0.001) than SFCS-coated curcumin in both low and high Her2/neu expressing breast cancer cells. Interestingly, the uptake of curcumin was highest for the high Her2/neu expressing breast cancer cells when delivered with a 10% w/v SF coating as compared to other formulations. In conclusion, SF-derived curcumin nanoparticles show higher efficacy against breast cancer cells and have the potential to treat in vivo breast tumors by local, sustained, and long-term therapeutic delivery as a biodegradable system.
International Journal of Nanomedicine 01/2009; 4:115-22. · 3.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Soft tissue loss presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a silk fibroin-chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose-derived stem cells (ASCs) in a murine soft tissue injury model. Green fluorescent protein (GFP)-labeled ASCs were seeded on SFCS scaffolds at a density of 1 x 10(5) ASCs per cm(2) for 48 hours and then suture-inlaid to a 6-mm, full-thickness skin defect in 6-week-old male athymic mice. Wound healing was tracked for 2 weeks by planimetry. Histology was evaluated at 2 and 4 weeks. Our data show that the extent of wound closure was significantly enhanced in the ASC-SFCS group versus SFCS and no-graft controls at postoperative day 8 (90% +/- 3% closure vs. 75% +/- 11% and 55% +/- 17%, respectively). Microvessel density at wound bed biopsy sites from 2 weeks postoperative was significantly higher in the ASC-SFCS group versus SFCS alone (7.5 +/- 1.1 vs. 5.1 +/- 1.0 vessels per high-power field). Engrafted stem cells were positive for the fibroblastic marker heat shock protein 47, smooth muscle actin, and von Willebrand factor at both 2 and 4 weeks. GFP-positive stem cells were also found to differentiate into epidermal epithelial cells at 4 weeks postoperative. In conclusion, human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound healing and show differentiation into fibrovascular, endothelial, and epithelial components of restored tissue.